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First published online May 20, 2005; 10.1104/pp.105.061408 Plant Physiology 138:990-997 (2005) © 2005 American Society of Plant Biologists Rapid Array Mapping of Circadian Clock and Developmental Mutations in Arabidopsis1Department of Cell Biology and Institute for Childhood and Neglected Diseases, Scripps Research Institute, La Jolla, California 92037 (S.P.H., F.G.H., J.L.P.-P., T.F.S., M.J.Y., S.A.K.); and Plant Biology and Genomic Analysis Laboratory, Salk Institute, La Jolla, California 92037 (J.O.B., S.J.L., J.R.E.)
Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F2 individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION 1, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays.
1 This work was supported by the National Institutes of Health (grant nos. GM56006 and GM67837 to S.A.K. and a Ruth L. Kirschstein National Research Service Award postdoctoral fellowship [GM071225] to S.P.H.), by the Department of Energy (grant no. DE–FG03–00ER15113), by the National Science Foundation (grant no. MCB–0213154 to J.R.E.), by the U.S. Department of Agriculture (National Research Initiative Competitive Grants Program postdoctoral fellowship to S.J.L.), and by the Helen Hay Whitney Foundation (fellowship to J.O.B.). F.G.H. is a Department of Energy-Energy Biosciences Fellow of the Life Sciences Research Foundation. This is manuscript number 17134–CB of the Scripps Research Institute. 2 These authors contributed equally to the paper. 3 Present address: Ifeva, Facultad de Agronomia, UBA, Av. San Martin 4453, 1417, Buenos Aires, Argentina. 4 Present address: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 97516. 5 Present address: Department of Evolution and Ecology, University of Chicago, Chicago, IL 60608. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.061408. * Corresponding author; e-mail ecker{at}salk.edu; fax 858–558–6379. Received February 16, 2005; returned for revision March 27, 2005; accepted April 13, 2005. This article has been cited by other articles:
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